There is an urgent need for new HIV-1 therapies targeting different steps of the viral replicative cycle to combat the growing prevalence of multidrug-resistant viruses and to reduce treatment toxicities. We demonstrated that the chemokine receptor CCR5 serves as a critical portal of HIV-1 entry by acting as a fusion coreceptor in conjunction with CD4, the primary receptor for HIV-1. CCR5 plays a central role in virus transmission and pathogenesis, and therefore represents an attractive target for new HIV-1 therapies. PRO 140 is a unique humanized CCR5 monoclonal antibody (mAb) that offers a novel therapeutic profile. Unlike small-molecule CCR5 antagonists under development, PRO 140 broadly and potently inhibits CCR5-mediated HIV-1 entry without blocking or otherwise dysregulating the natural activities of CCR5. In addition, PRO 140 has demonstrated favorable tolerability and pharmacokinetic profiles in an ongoing Phase la clinical trial in healthy volunteers. PRO 140 is clearly differentiated from small molecules in terms of its lack of CCR5 antagonism, non-overlapping patterns of viral resistance, antiviral synergy, excellent tolerability profile, and potential for infrequent (e.g., monthly) dosing. PRO 140 may therefore define a unique CCR5 inhibitor subclass, an issue that we will explore in Project 1. The highly innovative nature of this therapeutic approach is further underscored by the fact that no CCR5 inhibitor and no mAb to any target have been approved for HIV-1 therapy. The primary objective of Project 1 is to identify viral and host determinants of the antiviral activity of PRO 140 and small-molecule CCR5 antagonists, addressing issues of forced resistance, cross-resistance, and potential synergistic effects. Initial studies will compare PRO 140 and small molecules for their potency and breadth of antiviral activity in vitro. We will endeavor to correlate variations in potency with relevant viral and host factors. We will compare the evolution of HIV-1 resistance to PRO 140 and small molecules and determine the degree of cross-resistance. We will also examine whether PRO 140 can be usefully combined with small molecules and with HIV-1 inhibitors that have other mechanisms of action, and we will explore the molecular basis for any synergies observed. An important aspect of Project 1 is to correlate our in vitro findings with the clinical responses observed in the same patients treated with PRO 140 in Project 2. We will therefore perform a series of in vitro and ex vivo studies that are integrated with the PRO 140 clinical trials. This will enable us to directly test our hypotheses about the potential viral and host factors that influence the outcome of CCR5 therapy in humans. Our findings will be augmented by tropism, susceptibility and other analyses performed using complementary technologies in Project 3. Overall, Project 1 will provide basic insights into CCR5 inhibitor function and will lay the groundwork for deploying PRO 140 and small-molecule CCR5 antagonists in a manner that provides maximum therapeutic benefit to HIV-1 infected individuals.